Fusion pore formation in the influenza hemagglutinin (HA)- mediated fusion is a culmination of a multistep process, which involves low-pH triggered refolding of HA coupled in yet unknown way to rearrangement of membrane lipid bilayers. In the absence of the target membrane, low pH triggers irreversible inactivation of HA. We used biochemical and functional assays to study the low pH activation and inactivation of X31 and Japan strains of HA. Since HA inactivation by low pH pretreatment is much more profound and fast for X31 HA than for Japan HA, the latter has been thought to inactivate much slower than the former. Contrary to this interpretation, we found that the difference between X31 and Japan HA lays in the rates of HA activation rather than inactivation as thought before. For instance, 10-min application of pH 4.9 to HA-cells triggers activation of 80- 90% of X31 HA vs. 10-35% of Japan HA. For the few activated Japan HA molecules the inactivation rate was similar to that of X31 HA. These unexpected results argue against existence of a hypothetical activated intermediate conformation of HA, that is long-living and transient for Japan and X31 HA, respectively (Puri et al.,1990, J. Virol., 64, 3824) and formulate a new challenge to find the structural determinants of the rates of HA activation. Along with these studies on the conformational changes in the fusion protein, we have continued characterization of the membrane intermediates in the fusion pathway. Hemifusion, the linkage of contacting lipid monolayers of two membranes prior to the opening of a fusion pore, has been hypothesized to proceed through the formation of a stalk intermediate, a local and strongly bent connection between membranes. When the monolayers propensity to bend does not support the stalk (e.g. as it is when lysophosphatidylcholine, LPC, is added) hemifusion is inhibited. Short-chain alcohols (e.g. ethanol and methanol) are known to affect monolayer bending similarly to LPC. However we found these alcohols (1.2-1.6 w/w %) to promote rather than inhibit hemifusion between protein-free lipid bilayers. Promotion of membrane hemifusion by short-chain alcohol was also observed for HA-mediated cell-cell fusion. We propose that binding of short-chain alcohol to the surface of membranes promotes hemifusion by facilitating the transient breakage of the continuity of each of the contacting monolayers, which is required for their subsequent merger in the stalk intermediate. Our earlier finding that disparate fusion processes similarly depend on membrane lipid composition let us to hypothesize that stalk formation can be a pivotal event in ubiquitous biological fusion. Studies on the mechanism of stalk formation for protein-free bilayers and for fusion mediated by low-pH activated HA could give us important insights on this fundamental mechanism. - membrane fusion, influenza virus hemagglutinin, lipid monolayer, short- chain alcohol, liposome-planar bilayer fusion